Antisense oligonucleotide against human acetylcholinesterase (AChE) and uses thereof

ABSTRACT

The invention relates to an antisense oligonucleotide targeted to the coding region of the human acetylcholinesterase (AChE), which selectively suppresses the AChE-R isoform of the enzyme. The antisense oligonucleotide is intended for use in the treatment and/or prevention of neuromuscular disorders, preferably  myasthenia gravis.  In addition, it can penetrate the blood-brain barrier (BBB) and destroy AChE-R within central nervous system neurons, while also serving as a carrier to transport molecules across the BBB.

FIELD OF THE INVENTION

[0001] The present invention relates to a synthetic antisenseoligodeoxynucleotide targeted to the common coding domain of humanacetylcholinesterase (AChE) mRNA, and to pharmaceutical or medicalcompositions comprising the same, particularly for the treatment and/orprevention of a progressive neuromuscular disorder.

BACKGROUND OF THE INVENTION

[0002] All publications mentioned throughout this application are fullyincorporated herein by reference, including all references citedtherein.

[0003] Neuromuscular junctions (NMJ) are highly specialized,morphologically distinct, and well-characterized cholinergic synapses[Hall and Sanes (1993) Cell 72 Suppl., 99-121]. Chronic impairments inNMJ activity induce neuromuscular disorders characterized by progressivedeterioration of muscle structure and function. The molecular andcellular mechanisms leading from compromised NMJ activity to musclewasting have not been elucidated.

[0004] One such disorder is myasthenia gravis (MG), caused by a defectin neuromuscular transmission mediated by auto-antibodies that severelyreduce the number of functional post-synaptic muscle nicotinicacetylcholine receptors (nAChR) [Drachman D. G. (1994) N. Engl. J. Med.330, 1797-1810; Vincent A. (1999) Curr. Opin. Neurol. 12, 545-551]. MGis characterized by fluctuating muscle weakness that may be transientlyimproved by inhibitors of acetylcholinesterase (AChE) [Penn A. S. andRowland L. P. (1995) Myasthenia Gravis In: Meritt's Textbook ofNeurology, 9^(th) Edition, Williams and Wilkins, Baltimore, sectionXVII, 754-761]. The characteristic electrodiagnostic abnormality is aprogressive, rapid, decline in the amplitude of compound muscle actionpotentials (CMAP) evoked by repetitive nerve stimulation at 3 or 5 Hz.To date, the standard treatment for MG includes immunosuppressivetherapy combined with chronic administration of multiple daily doses ofperipheral AChE inhibitors such as pyridostigmine (Mestinon™). WhileAChE inhibitors effectively restore muscle performance in MG patients,their effects are short-lived, calling for the development of additionaleffective treatment.

[0005] Antisense technology offers an attractive, gene-based alternativeto conventional anti-cholinesterase therapeutics. Antisense technologyexploits the rules of Watson-Crick base pairing to design shortoligonucleotides, 15-25 residues in length, whose sequence iscomplementary to that of a target mRNA [Agrawal S. and Kandimalla E. R.(2000) Mol. Med. Today, 6, 72-81]. Stretches of double-stranded RNA,resulting from hybridization of the antisense oligonucleotide (ASON)with its target, activate RNAse H [Crooke S. T. (2000) Methods Enzymol.313, 3-45] and promote specific degradation of the duplex mRNA. Asantisense therapeutics target RNA rather than proteins, they offer thepotential to design highly specific drugs with effective concentrationsin the nanomolar range [Galyam N. et al. (2001) Antisense Nucleic AcidDrug Dev. 11, 51-57]. Phosphorothioated and 3′ terminally protected2′-O-methyl antisense oligonucleotides targeted to mouse AChE mRNA wereshown to be effective in blocking AChE expression in vitro in culturedhuman and rodent cells [Koenigsberger C. et al. (1997) J. Neurochem. 69,1389-1397; WO 98/26062; Grisaru D. et al. (2001) Mol. Med. 7, 93-105],and in vivo in brain [Shohami E. et al. (2000) J. Mol. Med. 78, 278-236;Cohen et al. (2002) Molecular Psychiatry, in press], muscle [Lev-LehmanE. et al. (2000) J. Mol. Neurosci. 14, 93-105] and bone marrow [Grisaruet al. (2001) ibid.].

[0006] The inventors have recently observed that treatment with theirreversible cholinesterase inhibitor diisopropylfluorophosphonate (DFP)induces overexpression of an otherwise rare, non-synaptic alternativesplicing variant of AChE, ACHE-R, in brain [Kaufer D. et al. (1998)Nature, 393, 373-377] and intestine [Shapira M. et al. (2000) Hum. Mol.Genet. 9, 1273-1282]. Muscles from animals treated with DFP alsooverexpressed AChE-R, accompanied by exaggerated neurite branching,disorganized wasting fibers and proliferation of NMJs. Partiallyprotected 2′-O-methyl antisense oligonucleotides targeted to mouse AChEmRNA suppressed feedback upregulation of AChE and amelioratedDFP-induced NMJ proliferation [Lev-Lehman et al. (2000) ibid.]. Theseobservations demonstrated that cholinergic stress elicits overexpressionof AChE-R in muscle and that antisense oligonucleotides can suppresssuch AChE-R excess and prevent its deleterious outcome.

[0007] As mentioned above, the characteristic electrodiagnosticabnormality is a progressive, rapid decline in the amplitude of muscleaction potentials evoked by repetitive nerve stimulation at 3 or 5 Hz.This myasthenic fatigue is caused by decrease in the number of AChRmolecules available at the post-synaptic site. Inhibiting anti-AChRantibodies are present in 85% to 90% of patients [Vincent, A. (1999) idibid].

[0008] Patients with MG, but not with congenital myasthenias due toother causes [Triggs et al. (1992) Muscle Nerve 15, 267-72], display atransient clinical response to AChE inhibitors such as edrophonium. Theavailable anti-AChE drugs are the first line of treatment, but mostpatients require further help. This includes drastic measures, such asplasma exchange, thymectomy and immunosuppression. Unfortunately, all ofthe currently employed MG drug regimens .are associated with deleteriouslong-term consequences. These include disturbance of neuromusculartransmission, exacerbation and induction of MG symptoms. Also, theotherwise safe use of common drugs such as anti-infectives,cardiovascular drugs, anticholinergics, anticonvulsants, antirheumaticsand others has been reported to worsen the symptoms of MG patients[Wittbrodt (1997) Arch. Intern. Med., 157, 399-408].

[0009] While the neuromuscular malfunctioning associated with MG can betransiently alleviated by systemic chronic administration of carbamateacetylcholinesterase (AChE) inhibitors (e.g. pyridostigmine), theinventors have found that pyridostigmine induces a feedback responseleading to excess AChE accumulation [Friedman et al. (1996) NatureMedicine 2, 1382-1385; Kaufer et al. (1998) id ibid; Meshorer, E. et al.(2002) Science 295, 508-12]. This suggested that the chronic use of suchinhibitors would modify the cholinergic balance in the patients'neuromuscular system and would require increased doses of these drugs;it also provided an explanation of the highly variable dose regimenemployed in MG patients; and it called for the development of analternative approach to suppress acetylcholine hydrolysis.

[0010] AChE-encoding RNA is subject to 3′ alternative splicing yieldingmRNAs encoding a “synaptic” (S) isoform, containing exons 1-4 and 6,also designated E6 mRNA herein, an “erythrocytic” (E) isoform,containing exons 1-6, also designated E5 mRNA herein, and the“readthrough” AChE-R derived from the 3′-unspliced transcript,containing exons 1-6 and the pseudo-intron 14, also designated 14 mRNAherein.

[0011] Transgenic mice overexpressing human AChE-S in spinal cordmotoneurons, but not in muscle, displayed progressive neuromotorimpairments that were associated with changes in NMJ ultrastructure[Andres, C. et al. (1997) Proc. Natl. Acad. Sci. USA 94, 8173-8178].However, it was not clear whether the moderate extent of overexpressedAChE in muscle was itself sufficient to mediate this severemyopathology. In rodent brain, the inventors found previously that bothtraumatic stress and cholinesterase inhibitors induce dramaticcalcium-dependent overexpression of AChE-R [Kaufer, et al. (1998) idibid.], associated with neuronal hypersensitivity to both cholinergicagonists and antagonists [Meshorer et al. (2002) id ibid].

[0012] Chronic AChE excess was found to cause progressive neuromotordeterioration in transgenic mice and amphibian embryos [Ben Aziz-Aloyaet al. (1993) Proc. Natl. Acad. Sci. USA, 90, 2471-2475; Seidman et al.(1994) J. Neurochem. 62, 1670-1681; Seidman, et al. (1995) Mol. Cell.Biol. 15, 2993-3002; Andres, C. et al. (1997) Proc. Natl. Acad. Sci. USA94, 8173-8178; Sternfeld et al. (1998) J. Neurosci. 18, 1240-1249].Also, myasthenic patients suffer acute crisis events, with a reportedaverage annual incidence of 2.5% [Berrouschot et al. (1997) Crit. CareMed. 25, 1228-35] associated with respiratory failure reminiscent ofanti-AChE intoxications.

[0013] In one approach, the prior art teaches that chemically protectedRNA aptamers capable of blocking the autoantibodies to the nicotinicAcetylcholine Receptor (nAChR) may be developed and used to treat MG.This approach has several drawbacks in that the RNA aptamers do not havethe amplification power characteristic of the RNAse-inducing antisenseagents and in that it fails to address the problem of the feedbackresponses in MG.

[0014] The present inventors have previously found that antisenseoligonucleotides against the common coding region of AChE are useful forsuppressing AChE production [WO 98/26062]. This publication also teachesthat antisense oligonucleotides against the human AChE are useful in thetreatment of memory deficiencies as observed in transgenic mice thatexpressed human AChE in their brain. The observed effects (see Table 4-5in WO 98/26062) are similar in their effect, yet considerably longer inthe duration of their action than the prior art AChE inhibitor tacrine(see FIG. 9B in WO 98/26062).

[0015] In view of the above, it is desirable to further improve thetreatment approaches for MG and other diseases involving impairment inneuromuscular transmission. The prior art treatment involving the use ofAChE inhibitors is afflicted with undesirable side effects because ofthe induction of AChE and neuromuscular impairments by such inhibitors;and because it is subject to variable efficacy under altered mentalstate (stress).

[0016] WO01/36627 teaches that morphological and functional changes inthe NMJ correlate with overexpression of a specific isoform of AChEmRNA, viz., the “readthrough” isoform containing the pseudo-intron I4 inthe mature mRNA. Said PCT application also shows that antisenseoligonucleotides directed to the common coding region of AChE may beused to specifically destroy AChE-R mRNA, and that AChE antisense agentsare by far superior to conventional AChE enzyme inhibitor drugs in thetreatment of neuromuscular disorders. The superiority of these antisenseagents may be due to the fact that conventional enzyme inhibitorsactively induce I4 AChE mRNA overexpression. According to the teachingsof WO01/36627, this may lead to detrimental changes in the NMJ. Thisconsequence of treatment may be entirely avoided by using the antisenseagents of WO01/36627.

[0017] The Blood-Brain Barrier (BBB) maintains a homeostatic environmentin the central nervous system (CNS). The capillaries that supply theblood to the brain have tight junctions which block the passage of mostmolecules through the capillary endothelial membranes. While themembranes do allow passage of lipid soluble materials, water solublematerials do not generally pass through the BBB. Mediated transportmechanisms exist to transport the water soluble glucose and essentialamino acids through the BBB. Active support mechanisms remove moleculeswhich become in excess, such as potassium, from the brain [for generalreview see Betz et al., Blood-Brain-Cerebrospinal Fluid Barriers,Chapter 32, in Basic Neurochemistry, 5^(th) ed., Eds Siegel, AlbersAgranoff, Molinoff, pp.681-701; Goldstein and Betz (1986) ScientificAmerican, September, pp. 74-83].

[0018] The BBB impedes the delivery of drugs to the CNS. Methods havebeen designed to deliver needed drugs such as direct delivery within theCNS by intrathecal delivery can be used with, for example, an Omayareservoir. U.S. Pat. No. 5,455,044 provides for the use of a dispersionsystem for CNS delivery [for description of other CNS deliverymechanisms, see U.S. Pat. No. 5,558,852, Betz et al., ibid., andGoldstein and Betz, ibid.]. Tavitan et al. [Tavitan et al. (1998) NatMed 4(4): 467-71] observed that 2′-O-methyl oligonucleotides are able topenetrate into the brain. Other systems make use of specially designeddrugs that utilize the structure and function of the BBB itself todeliver the drugs, for example by designing lipid soluble drugs or bycoupling to peptides that can penetrate the BBB.

[0019] It has been shown that stress affects the permeability of the BBB[Sharma H. S. et al. (1992) Prog. Brain Res. 91, 189-196; Ben-Nathan D.et al. (1991) Life Sci. 489, 1493-1500]. Further, in mammals, acutestress elicits a rapid, transient increase in released acetylcholinewith a corresponding phase of increased neuronal excitability [ImperatoA. et al. (1991) Brain Res. 538, 111-117]. It has been previouslyobserved by the present inventors that the AChE-R isoform and the I4peptide of AChE can act as stress mimicking agents and rupture the BBB.These findings formed the basis for PCT application WO98/22132, thecontents of which are fully incorporated herein by reference. WO98/22132relates to compositions for facilitating the passage of compoundsthrough the BBB, comprising the AChE-R splice variant and/or the peptideI4.

[0020] In search for an antisense oligonucleotide targeted against adomain of the human AChE, which may be particularly acceptable in humantherapy, the inventors have now found, and this is an object of thepresent invention, that a synthetic antisense oligodeoxynucleotidehaving the nucleotide sequence: 5′-CTGCCACGTTCTCCTGCACC-3′, hereindesignated SEQ ID NO:1, is not only useful in selectively suppressingthe production of the AChE-R isoform, but also possesses cross-speciesspecificity, which enables its use in rodent animal models of variousdiseases and, moreover, remarkably appears to penetrate the BBB, and maythus be useful in treatment of diseases of the central nervous system,alone or in combination with other therapeutic agents. The finding thatthe novel antisense of the invention can penetrate the BBB wasunexpected, particularly in view of the expectation that the BBB wouldbe impermeable to large polar molecules.

[0021] The application of antisense technology to the treatment ofnervous system disorders has, until recently, been considered to belimited by the lack of adequate systems for delivering oligonucleotidesto the brain. Nevertheless, several attempts have been made tocircumvent this difficulty [reviewed in Seidman S. et al. (1999)Antisense Nucl. Acid Drug Devel, 9, 333-340]. Access of chemical agentscirculating in the blood to the interstitial spaces of the brain isrestricted by the biomechanical barrier known as the BBB. The stronganionic character of the phosphodiester backbone makes oligonucleotidesespecially poor at crossing the BBB. In vivo pharmacokinetic studieshave demonstrated that less than 0.01% of a systemically injected doseof a phosphorothioate antisense oligonucleotide may reach the brain,where its residence time may be as little as 60 min. A research solutionto this problem in the laboratory is direct bypass of the BBB byintracranial injection of oligonucleotides. Using published stereotacticcoordinates for both rats and mice, oligonucleotides can be delivered bysingle injections, by repeated administration through an implantedcannula, or by continuous infusion using an osmotic mini-pump such asAlzet (Alza, Palo Alto, Calif.). Oligonucleotides can either bedelivered into the CSF or directly into the brain region of interest. Ingeneral, oligonucleotides are considered to remain relatively localizedfollowing intraparenchymal administration. Thus, a single injection of24 μg of an antisense oligonucleotide targeted to the cAMP-responseelement (CREB) into rat amygdala was reported to diffuse only 0.72±0.04μl around the injection site, exerting region-specific effects onconditioned taste aversion (CTA). Injection of the same oligonucleotideinto the basal ganglia 2 mm above the amygdala had no effect on CTA.Similarly, specific effects on behavior were reported following theinjection of antisense oligonucleotides against the stress-associatedtranscription factor c-fos into the medial frontal cortex (singleadministration; 10 μg), following delivery of oligonucleotides againstthe neurotransmitter-synthesizing enzyme glutamate decarboxylase intothe ventromedial hypothalamus (single administration; 1 μg), andfollowing 5 days continuous infusion of oligonucleotides targeted tomRNA encoding the cAMP-responsive transcription factor CREB into thelocus coeruleus (20 μg/day). It was further reported that widedistribution of oligonucleotides in the brain (up to 443 μl around thesite of injection after 48 hrs) could be achieved by direct, high-flowintraparenchymal microinfusion. In that case, the average tissueconcentration of oligonucleotide was calculated to be between 3-15μM—well within what is considered physiologically significant. Regardinguptake into neurons, it was shown that neurons in the striatum of ratspreferentially take up oligonucleotides compared to glia. Despite thegeneral retention of oligonucleotides around the injection site reportedin that study, some signal was observed to be transported alongprojection pathways to distant sites. However, to be effectivetherapeutically, oligonucleotides should be prepared in a way that wouldenable their stability and free penetrance into the central nervoussystem following intravenous injection, or yet more preferably,following oral administration. Thus, the present invention is aimed at anovel, preferably nuclease protected antisense oligodeoxynucleotidetargeted to the common coding domain of human AChE, which selectivelysuppresses the production of AChE-R, with rapid and long-lastingclinical improvements in muscle function, which possesses cross-speciesspecificity and can penetrate the BBB and destroy AChE-R mRNA withincentral nervous system neurons.

SUMMARY OF THE INVENTION

[0022] The invention relates to a pharmaceutical or medical compositionfor the treatment and/or prevention of a progressive neuromusculardisorder, comprising as active ingredient a synthetic antisenseoligodeoxynucleotide targeted against human AChE mRNA having thenucleotide sequence:

5′ CTGCCACGTTCTCCTGCACC 3′  (SEQ ID NO:1).

[0023] The antisense oligonucleotide preferably causes preferentialdestruction of AChE-R mRNA, possesses cross-species specificity, wasdemonstrated to cause no toxicity in rodents or primates, and canpenetrate the BBB in primates (monkeys) via both i.v. and p.o.administration routes.

[0024] In a preferred embodiment, the synthetic antisenseoligodeoxynucleotide having the nucleotide sequence designated SEQ IDNO:1 is nuclease resistant. The nuclease resistance may be achieved bymodifying the antisense oligodeoxynucleotide of the invention so that itcomprises partially unsaturated aliphatic hydrocarbon chain and one ormore polar or charged groups including carboxylic acid groups, estergroups, and alcohol groups.

[0025] In particular embodiments, the nuclease resistant antisenseoligodeoxynucleotide of the invention has at least one of the last three3′-terminus nucleotides is 2′-O-methylated, preferably the last three3′-terminus nucleotides are 2′-O-methylated. Alternatively, the nucleaseresistant antisense oligodeoxynucleotide of the invention may have atleast one of the last 3′-terminus nucleotides fluoridated. Stillalternatively, the nuclease resistant antisense oligodeoxynucleotide ofthe invention has phosphorothioate bonds linking between at least two ofthe last 3′-terminus nucleotide bases, preferably has phosphorothioatebonds linking between the last four 3′-terminal nucleotide bases. Stillalternatively, nuclease resistance may be achieved by the syntheticnuclease resistant antisense oligodeoxynucleotide of the inventionhaving a nucleotide loop forming sequence at the 3′-terminus, forexample a 9-nucleotide loop having the nucleotide sequence CGCGAAGCG(SEQ ID NO:2).

[0026] The synthetic nuclease resistant antisense oligodeoxynucleotideof the invention is capable of selectively modulating mammalian AChEproduction, particularly selectively modulating primate AChE productionin neurons residing in the central nervous system, including human AChEof interneurons.

[0027] In a further aspect, the invention relates to a pharmaceuticalcomposition comprising an antisense oligodeoxynucleotide of theinvention, and optionally further comprising pharmaceutically acceptableadjuvant, carrier or diluent.

[0028] In a preferred embodiment, the pharmaceutical composition of theinvention comprises an antisense oligodeoxynucleotide of SEQ ID NO:1,which is 2′-O-methylated on at least one, preferably the three last3′-terminus nucleotides.

[0029] The pharmaceutical composition of the invention is useful in thetreatment and/or prevention of a progressive neuromuscular disorder, forimproving stamina and/or for use in decreasing chronic muscle fatigue.

[0030] The pharmaceutical composition of the invention may be for a oncedaily use by a patient of a dosage between about 0.001 μg/g and about 50μg/g of active ingredient, preferably a dosage of active ingredient ofabout 0.01 to about 5.0 μg/g, more preferably a dosage of activeingredient of about 0.15 to about 0.5 μg/g.

[0031] The pharmaceutical composition of the invention is particularlyintended for use in treating or preventing a progressive neuromusculardisorder, wherein said disorder is associated with an excess of AChEmRNA or protein. Such a disorder may be, for example, a progressiveneuromuscular disorder, wherein said disorder is associated with anexcess of AChE-R mRNA.

[0032] The pharmaceutical composition of the invention is thusparticularly suitable for treating or preventing a progressiveneuromuscular disorder, wherein said disorder is associated withimpairment of cholinergic transmission.

[0033] Of particular interest are pharmaceutical compositions for thetreatment of a progressive neuromuscular disorder, wherein said disorderinvolves muscle distortion, muscle re-innervation or NMJ abnormalities,for example myasthenia gravis, Eaton-Lambert disease, musculardystrophy, amyotrophic lateral sclerosis, post-traumatic stress disorder(PTSD), multiple sclerosis, dystonia, post-stroke sclerosis, post-injurymuscle damage, post-surgery paralysis, excessive re-innervation, andpost-exposure to AChE inhibitors.

[0034] The pharmaceutical composition of the invention is also useful inimproving stamina in physical exercise or in decreasing muscle fatigue.

[0035] In addition, the invention relates to a pharmaceuticalcomposition comprising an antisense oligodeoxynucleotide as denoted bySEQ ID NO:1, for facilitating passage of compounds through the BBB,optionally further comprising additional pharmaceutically active agentand/or pharmaceutically acceptable adjuvant, carrier or diluent. Theadditional pharmaceutically active agent is a compound to be transportedthrough the BBB, wherein said compound may be contrast agents used forcentral nervous system imaging, agents that function to block theeffects of abused drugs, antibiotics, chemotherapeutic drugs and vectorsto be used in gene therapy. This composition would function primarily bysuppressing the production of AChE-R, which is apparently involved inBBB maintenance.

[0036] The invention will be described in more detail in the followingdetailed description and on hand of the following figures.

BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1 Human AChE mRNA [GenBank Accession No. M55040; Soreq etal., Proc. Natl. Acad. Sci. USA 87(24), 9688-9692 (1990)], and humanEN101 (hEN101, SEQ ID NO:1), targeted at nucleotides 795-5′ to 3′-814(shaded) of the coding sequence.

[0038] FIGS. 2A-B Representation of various physical and chemicalproperties of the human EN101 (SEQ ID NO:1).

[0039]FIG. 2A: Internal structure that is expected for theoligonucleotide, and an estimate of the energy (in kcal/mol) required todisrupt that structure.

[0040]FIG. 2B: Base composition and the predicted melting temperature ofits hybrid with the complementary mRNA.

[0041]FIG. 3 Mouse AChE mRNA [GenBank Accession No. X56518; Rachinsky etal. (1990) Neuron 5(3), 317-327], and mouse EN101 (mEN101, SEQ ID NO:3),targeted at nucleotides 639-5′ to 3-658 (shaded) of the coding sequence.

[0042] FIGS. 4A-B Representation of various physical and chemicalproperties of the mouse EN101 (SEQ ID NO:3).

[0043]FIG. 4A: Internal structure that is expected for theoligonucleotide, and an estimate of the energy (in kcal/mol) required todisrupt that structure.

[0044]FIG. 4B: Base composition and the predicted melting temperature ofits hybrid with the complementary mRNA.

[0045]FIG. 5 Rat AChE mRNA (partial, 2066 nucleotides) [GenBankAccession No. S50879; Legay et al. (1993), J. Neurochem. 60(1),337-346], and the rat EN102 (rEN102, SEQ ID NO:5), targeted atnucleotides 51-5′ to 3′-70 (shaded) and rat EN101 (rEN101, SEQ ID NO:4),targeted at nucleotides 639-5′ to 3′-658 (shaded) of the codingsequence.

[0046] FIGS. 6A-B Representation of various physical and chemicalproperties of the rat EN101 (SEQ ID NO:4).

[0047]FIG. 6A: Internal structure that is expected for theoligonucleotide, and an estimate of the energy (in kcal/mol) required todisrupt that structure.

[0048]FIG. 6B: Base composition and the predicted melting temperature ofits hybrid with the complementary mRNA.

[0049]FIG. 7 Immunoreactive AChE-R in EAMG rats.

[0050] Separation of rat serum was performed in non-denaturingpolyacrylamide gel, and the gel tested for immunoreactive AChE-R. AnEAMG rat had considerably higher level of the rapidly migrating rRvariant than a control rat.

[0051] Abbreviations: electroph., electrophoresis; cont., control.

[0052] FIGS. 8A-C Excess AChE-R expression in muscles of EAMG rats.

[0053]FIG. 8A: AChE mRNA transcripts expressed in muscle. Shown is thestress-responding mammalian ACHE gene, with a functional glucocorticoidresponse element (GRE) in its distal enhancer, and its two mRNAtranscripts expressed in muscle. Note that exon 6 is unique to thesynaptic transcript AChE-S, whereas, pseudo-intron 4′ is expressed onlyin the stress induced AChE-R mRNA. Antibodies targeted to thepseudo-intron 4′-derived C-terminal peptide served to detect the AChE-Rprotein, and cRNA probes to exon 6 and pseudo-intron 4′ label the twotranscripts (asterisks).

[0054]FIG. 8B: Depleted nAChR and excess AChE-R in EAMG muscles. Shownis immunohistochemical staining of paraffin-embedded sections of tricepsmuscle from normal or EAMG rats treated with the inert inverse(r-invEN102) oligonucleotides, similar to those of untreated rats.Staining was with polyclonal rabbit antibodies to nAChR (1,2) and AChE-R(3,4). Immunopositive areas are stained red. Note that the AChE-Rprotein was prominently elevated and nAChR dramatically reduced in EAMG.In situ hybridization with probes specific for AChE-R or -S mRNAsyielded red stained RNA, with DAPI (white) used to visualize cellnuclei. Note the prominent sub-nuclear accumulation of AChE-R mRNA inpreparations from EAMG, but not control animals (5,6). AChE-S mRNAdisplayed punctuated expression in subnuclear areas in both control andEAMG rats (7,8).

[0055]FIG. 8C: rEN101 treatment. In EAMG rats, EN101 reduced levels ofAChE-R (1,2) and AChE-R mRNA (5,6), but did not affect nAChR (3,4) orAChE-S mRNA (7,8), as compared to rats treated with the inverse sequence(see FIG. 8B, above.).

[0056] Abbreviations: healt., healthy; r., rat; prot., protein.

[0057] FIGS. 9A-D Normalized EAMG muscle electrophysiology undersuppression of AChE-R.

[0058]FIG. 9A: Immunoreactive AChE-R was detected, as in FIG. 7B, in theserum of healthy and severely affected EAMG rats, treated with rEN101 orr-invEN102, and the densities of the bands are represented in the bargraph.

[0059]FIG. 9B: Animals (at least 6 rats in each group) were treated witha single i.p. injection (75 μg/kg) of the AChE inhibitor neostigmine,and the CMAP ratio relative to the baseline was measured. The averageCMAP ratio of EAMG rats included in the study prior to treatment was87±2.5% of first depolarization, and average CMAP ratio inrEN101-treated animals was 107.4±3.8% (inset).

[0060]FIG. 9C: Animals (at least 6 rats in each group) were treated withvarious doses of rEN101. The treatment (doses between 10-500 μg/Kg)restored the CMAP decline for up to 72 h. Note that higher dosesconferred increasingly longer-lasting relief.

[0061]FIG. 9D: Dose response curse. CMAP responses at each time wereplotted as a function of EN101 concentration. Note that at 1 and 5 hthere are clearly two effects, a steep increase dependent on a low EN101concentration (IC₅₀<10 μg/kg), superimposed on a much lower-affinityeffect that persists much longer.

[0062] Abbreviations: ser., serum; t., time; dep., dependence; neostig.,neostigmine; resp., response; h., hours; perc., percent; cont., control;rat., ratio; bas., baseline.

[0063]FIG. 10 Rat EN101 (SEQ ID NO:4) improves stamina in myasthenicrats.

[0064] Experimental autoimmune myasthenic gravis (EAMG) rats withvarying severity of clinical symptoms and healthy Lewis rats wereprodded to run on an electrically powered treadmill (25 m/min, inset)until visibly fatigued. Presented is the average time (sec.±SEM) ratswere able to run before and 24 h following i.v. administration of 250μg/kg rEN101. Note that running time for EAMG rats decreased withdisease severity, and increased for each group treated with rEN101.

[0065] Abbreviations: trml., treadmill; treat., treatment; h., hour;clin., clinical; stat., status; run., running; t., time; sec., seconds.

[0066] FIGS. 11A-B Stable reversal of declining CMAP response in EAMGrats treated orally with rEN101.

[0067] EAMG rats received rEN101 once daily for up to 4 days byintravenous injection (25 μg/kg) or via oral gavage (50 μg/kg), orpyridostigmine (1000 μg/kg) by oral gavage. The CMAP ratio wasdetermined 1 and 5 h following the first drug administration and thenevery 24 h, prior to the administration of the subsequent dose.

[0068]FIG. 11A: Single dose. Orally administered pyridostigmine (n=4)and rEN101 (n=8) relieved the declining CMAP responses within 1 h. 24 hfollowing administration of pyridostigmine, CMAP ratios in muscles oftreated rats returned to the declining baseline. In contrast, no declinewas detected in rats treated with rEN101.

[0069]FIG. 11B: Repeated daily doses. The graph depicts the equivalentimprovement in muscle function elicited by oral (50 μg/kg, n=8) ascompared to i.v. (25 μg/kg, n=4) administration of rEN101. Note thatrepeated administration of rEN101 conferred stable, long-termalleviation of CMAP declines. Repeated daily administration ofpyridostigmine at 24 h intervals yielded considerably shorter CMAPimprovements than those obtained with EN101, decreasing back to thedeclining baseline prior to the next dose.

[0070] Abbreviations: sing., single; dos., dose; rep., repeated; d.,daily; pyridostig., pyridostigmine; h., hours; rat., ratio; bas.,baseline; ab., above.

[0071] FIGS. 12A-C: Long-term rEN101 treatment changes the course ofEAMG.

[0072]FIG. 12A: Survival. A greater fraction of animals treated oncedaily with rEN101 (50 μg/Kg, daily, p.o.) survived than those treatedwith pyridostigmine (1000 μg/Kg) despite their similarly poor initialstatus and initial number of animals in each group.

[0073]FIG. 12B: Clinical status. Shown are average values for theclinical status (as defined in Experimental Procedures) of survivinganimals from each of the treated groups. Note increasing severity ofdisease in saline- and pyridostigmine-treated animals, as compared tothe improved status of rEN101-treated animals.

[0074]FIG. 12C: Stamina. Shown are average running times in sec. forrEN101- and pyridostigmine-treated animals. Note that before treatment,EAMG rats performed as severely sick animals (clinical status 4).

[0075] Abbreviations: surv., survival; clin., clinical; stat., status;stam., stamina; an., animals; al., alive; sc., score; run., running; t.,time; sec., seconds; w., weeks; sal., saline; pyridostig.,pyridostigmine.

[0076]FIG. 13 Proposed model for EN101 activity

[0077] At the neuromuscular junction, acetylcholine (ACh) released fromthe motoneuron terminal (top) into the synaptic cleft travels towardsthe muscle postsynaptic membrane (below). There, it interacts with nAChRto initiate an inward ion current and elicit muscle action potentials.ACh is subsequently hydrolyzed by synapse-bound AChE-S. Subsynapticmuscle nuclei (ellipses) produce, in addition to the primary AChE-S mRNAtranscript, the normally rare AChE-R mRNA with its alternative 3′-end.This transcript translates into soluble, secretory AChE-R monomers.Myasthenic autoimmune antibodies toward nAChR block the initiation ofaction potentials, mimicking an ACh-deficient state. The cholinergicimbalance results in AChE-R accumulation that enhances ACh destruction,leading to muscle fatigue. Chemical anticholinesterases (indentedcircles) non-selectively block both AChE-S and AChE-R, which transientlyincreases ACh levels, yet further intensifies AChE-R overproduction. Incontrast, the antisense agent EN101 selectively induces AChE-R mRNAdestruction, preventing AChE-R synthesis while maintaining AChE-S andsustaining normal neuromuscular transmission.

[0078]FIG. 14 Dose-dependent HEN101 suppression of neuronal AChE-R mRNA,but not of AChE-S mRNA.

[0079] Shown are representative fields from spinal cord sections ofhEN101-treated monkeys following in situ hybridization with AChE-R orAChE-S cRNA probes. Note that AChE-R mRNA labeling decreased, but AChE-SmRNA levels appeared unchanged. An increasing dose of o.g.-administeredhEN101 suppressed AChE-R mRNA more effectively, suggestingdose-dependence. Administration of the higher dose via i.v. appearedmore effective than the o.g. route.

[0080] Abbreviations: d., day.

[0081]FIG. 15 hEN101-suppression of neuronal AChE-R mRNA levels is celltype-specific.

[0082] Spinal cord neurons from hematoxylin-eosin stained monkeysections were divided by size into cells with perikaryal diameters of<40, 40 to 70 and >70 μm. The percent of cells within each size groupthat were positively labeled for AChE-R mRNA was recorded in 5 differentfields of 1 mm² each, for each hEN101 treatment. Note that hEN101effectiveness was apparently highest in the relatively smallinterneurons, and lowest in the largest motoneurons.

[0083] Abbreviations: pos., positive; cel., cell; siz., size; gr.,group; bo., body; diam., diameter; nv., naive.

[0084]FIG. 16 Shown are levels of hydrolyzed acetylthiocholine,indicating acetycholinesterase activity in the plasma of cynomolgousmonkeys treated i.v. for two consecutive days with 150 or 500 μg/kghEN101 or with orally administered 500 μg/kg hEN101.

[0085]FIG. 16A: Total activity.

[0086]FIG. 16B: Activity under 5×10⁻⁵ M of iso-OMPA (AChE). Noteinjection-induced increases in enzyme activity and AS-ON reductions.

[0087] Abbreviations: t., time; fol., following; treat., treatment;hrs., hours.

[0088]FIG. 17 Effect of rEN101 on AChE-R mRNA in rat spinal cordneurons. The presence of AChE-R mRNA-positive cells was determined inspinal cord section of rats that had been treated for 7 days with rEN101(500 μg/kg, i.v., daily).

[0089] Abbreviations: cont., control.

[0090]FIG. 18 hEN101 alleviates ptosis in MG patients. Photographs show:before hEN101 treatment (upper panel), on 10 Mestinon®/day, 600 mg;during hEN101 treatment (middle panel), 500 μg/kg, 2-day treatment; and4 weeks after hEN101 treatment (lower panel), back to Mestinon®treatment.

[0091]FIG. 19 Efficacy of oral hEN101 in MG patients. Graph shows meanchange from baseline of one patient in total grade. This patient was a56 year old male, myasthenic for 29 years, who was treated withpyridostigmine and had a baseline of QMG=6. He returned to thepyridostigmine treatment 72 hours after the last hEN101 dose.

[0092]FIG. 20 Improvement in Total QMG Score. Graph shows the meanpercentage improvement (plus standard deviation) in total QMG score ofall patients, from the baseline value until day 6 of treatment.

[0093] FIGS. 21A-B hEN101 improves myasthenic status.

[0094]FIG. 21A Graph shows QMG score of all the patients included in theclinical trial.

[0095]FIG. 21B Graph shows mean change from baseline in QMG score.

DETAILED DESCRIPTION OF THE INVENTION

[0096] For the purposes of clarity, the following abbreviations andterms are defined herein:

[0097] AChE: acetylcholinesterase

[0098] ACHE-R: acetylcholinesterase, “readthrough” variant or isoform,its mRNA includes pseudo-intron I4

[0099] AChE-S: acetylcholinesterase, synaptic variant or isoform

[0100] AS-ON: antisense oligonucleotide

[0101] BBB: blood-brain barrier

[0102] CMAP: compound muscle action potential

[0103] CNS: central nervous system

[0104] EAMG rat: rats wherein experimental autoimmune myasthenia gravishas been induced

[0105] EN101: may also be referred as AS3; antisense oligonucleotidetargeted against human, rat or mouse (hEN101, rEN101 or mEN101,respectively) AChE mRNA

[0106] EN102: may also be referred as AS1, antisense oligonucleotidetargeted against AChE mRNA, at a different region than EN101

[0107] MG: myasthenia gravis, a neuromuscular junction disease

[0108] i.v.: intravenous

[0109] o.g.: oral gavage

[0110] p.o.: per os

[0111] Antisense oligonucleotide: A nucleotide comprising essentially areverse complementary sequence to a sequence of AChE mRNA. Thenucleotide is preferably an oligodeoxynucleotide, but alsoribonucleotides or nucleotide analogues, or mixtures thereof, arecontemplated by the invention. The antisense oligonucleotide may bemodified in order to enhance the nuclease resistance thereof, to improveits membrane crossing capability, or both. The antisense oligonucleotidemay be linear, or may comprise a secondary structure. It may alsocomprise enzymatic activity, such as ribozyme activity.

[0112] Progressive neuromuscular disorder: A disorder or conditionassociated with excess AChE mRNA or protein production, characterized bychanges in the morphology of the NMJ and impairment in neuromusculartransmission. The neuromuscular disorder may involve muscle distortion,muscle re-innervation or NMJ abnormalities. More preferably, theprogressive neuromuscular disorder is myasthenia gravis, musculardystrophy, multiple sclerosis, amyotrophic lateral sclerosis,post-traumatic stress disorder (PTSD), or dystonia.

[0113] The present invention relates to a novel antisenseoligodeoxynucleotide substantially as denoted by SEQ ID NO:1, alsodesignated herein as hEN101.

[0114] In addition to the part of the sequence which is complementary toAChE sequence, the antisense oligonucleotide of the invention may alsocomprise RNA sequences with enzymatic nucleolytic activity, or may belinked to such sequences. Preferred nucleolytic sequences are ribozymesequences, which were shown to specifically interact with mRNAtranscripts. They are ribonucleic acid sequences, including RNase activesites flanked by antisense oligonucleotides [Haseloff and Gerlach (1988)Nature 3, p. 585, Sarver et al. (1990) Science 247, p. 1222]. Preferredribozymes are hammerhead ribozymes [Conaty et al. (1999) Nucleic AcidsRes. 27, 2400-2407; and Xu et al. (1999) Endocrinology, 140, 2134-44].Another preferred ribozyme is the hairpin ribozyme structure, e.g., asderived from tobacco ringspot virus satellite RNA [see Perez-Ruiz (1999)Antisense Nucleic Acid Drug Dev., 9, 33-42].

[0115] The novel antisense oligodeoxynucleotide of the inventioncorresponds to the reverse complement of human AChE mRNA sequence, fromnucleotide 795-5′ to nucleotide 3′-814 (FIG. 1). Prior work by thepresent inventors has demonstrated the usefulness of antisenseoligonucleotide in suppressing AChE production and in the treatment ofmemory deficiency. In said prior work, a number of AChE antisenseoligonucleotides have been disclosed. Said prior work further disclosesdesirable features of such antisense oligonucleotides and possiblemodifications thereof, such as nuclease resistance, modifications toenhance membrane transport of oligonucleotides, and the like. Said priorwork, e.g. WO 98/26026, is therefore incorporated herein in its entiretyby reference. In another publication, the present inventors describe therole of antisense oligonucleotides in the treatment of a variety ofneurodegenerative diseases [Seidman, S. et al., Antisense Res. Nucl.Acids Drug Devel. 9, 333-340 (1999)].

[0116] The antisense oligodeoxynucleotide of the invention is preferablynuclease resistant. There are a number of modifications that impartnuclease resistance to a given oligonucleotide. Reference is made to WO98/26062, which publication discloses that oligonucleotides may be madenuclease resistant e.g., by replacing phosphodiester internucleotidebonds with phosphorothioate bonds, replacing the 2′-hydroxy group of oneor more nucleotides by 2′-O-methyl groups, or adding a nucleotidesequence capable of forming a loop structure under physiologicalconditions to the 3′ end of the antisense oligonucleotide sequence. Anexample for a loop forming structure is the sequence 5′ CGCGAAGCG (SEQID NO:2), which may be added to the 3′ end of a given antisenseoligonucleotide to impart nuclease resistance thereon.

[0117] The cells on which the antisense oligonucleotide of the inventionexerts its effects are preferably muscle cells and cells of the NMJ,including the nerve axons and endplate structures.

[0118] Using the antisense oligonucleotides according to the invention,it is expected that AChE-R amount and AChE-R mRNA levels are reduced incentral nervous system neurons by at least about 30%, preferably by atleast about 40%, and more preferably by at least about 50%, within 24 hrof the treatment, and by about 80% under repeated treatment. Thisreduction was shown by fluorescent in situ hybridization (FISH) andimmune labeling and its effectiveness was confirmed by electrophysiologyand tread mill tests. It exceeded by far all previous reports of AS-ONdestruction of AChE-R mRNA in other cells and tissues.

[0119] In yet another embodiment of the invention, the preferredtreatment window of candidate oligonucleotides is evaluated by FISH. Thetechnique of in situ hybridization is well known to the man of skill inthe art, and is described e.g., In situ Hybridization, Wilkinson, D. G.(Ed.) ISBN: 0199633274; In situ Hybridization for the Brain, Wisden W.,Morris B. J. (Eds.), ISBN: 0127599207, PCR in situ Hybridization: APractical Approach (Practical Approach Series 186), Herrington C. S.,John O'Leary J., (Eds.) ISBN:019963632X. Detailed protocols relating toin situ hybridization using non-radioactively labeled probes areavailable from Microsynth GmbH (Balgach, Switzerland).

[0120] Labeled AChE-R cRNA sequences may be used as probes for in situhybridization. The ACHE cRNA probe preferably comprises I4 pseudo-intronsequences.

[0121] In a preferred embodiment of the invention, the AChE mRNAdetermination is carried out by using in situ RT-PCR, which technique isdescribed, e.g., in the above-mentioned references, see also PCR in situhybridization: Protocols and Applications, 3rd ed., by Nuovo, G. J.Lippincott, Raven Press, New York (1996).

[0122] Phosphorothioate-modified oligonucleotides are generally regardedas safe and free of side effects. Peng et al. teach that undesired invivo side effects of phosphorothioate antisense oligonucleotides may bereduced when using a mixed phosphodiester-phosphorothioate backbone. Theantisense oligonucleotides of the present invention have been found tobe effective as partially phosphorothioates and yet more effective aspartially 2′-O-methyl protected oligonucleotides. WO 98/26062 teachesthat AChE antisense oligonucleotides containing three phosphorothioatebonds out of about twenty internucleotide bonds are generally safe touse in concentrations of between about 1 and 10 μM. However, forlong-term applications, oligonucleotides that do not release toxicgroups when degraded may be preferred. These include 2′-O-methylprotected oligonucleotides, but not phosphorothioate oligonucleotides. Afurther advantage of 2′-O-methyl protection over phosphorothioateprotection is the reduced amount of oligonucleotide that is required forAChE suppression. This difference is thought to be related to theimproved stability of the duplexes obtained when the 2′-O-methylprotected oligonucleotides are used [Lesnik, E. A. & Freier, S. M.,Biochemistry 37, 6991-7, (1998)]. An alternative explanation for thegreater potency of the 2′-O-methyl oligonucleotides is that thismodification may facilitate penetration of the oligonucleotide chainthrough the cell membrane. A further advantage of 2′-O-methyl protectionis the better protection against nuclease-mediated degradation that itconfers, thus extending the useful life time of antisenseoligonucleotides protected in this way.

[0123] In accordance with the invention, the dosage of the antisenseoligodeoxynucleotide is about 0.001 to 50 μg oligonucleotide per gram ofbody weight of the treated animal. Preferably, the dosage is about 0.01to about 5.0 μg/g. More preferably, the dosage is between about 0.05 toabout 0.7 μg/g. Thus, the optimal dose range is between 50-500 μg/kg ofbody weight of the treated subject, for rats, monkeys and also humans.

[0124] The antisense oligonucleotide of the invention is provided foruse in the treatment of a disorder that involves excessive AChE mRNAproduction.

[0125] The disorder is preferably a disorder involving functional andmorphological changes in the NMJ.

[0126] The progressive neuromuscular disorder preferably involvesoverexpression of AChE-R mRNA.

[0127] More preferably, the disorder is selected from, but not limitedto, multiple sclerosis, PTSD, myasthenia gravis,muscular dystrophy,amyotrophic lateral sclerosis, dystonia, muscle distortion, musclere-innervation or excessive muscle innervation.

[0128] The excessive muscle innervation is selected preferably from, butnot limited to, excessive innervation after trauma, preferably afteramputation.

[0129] In one aspect, the invention relates to a pharmaceuticalcomposition for the treatment and/or prevention of a progressiveneuromuscular disorder, for improving stamina in physical exerciseand/or for use in decreasing chronic muscle fatigue, comprising asactive ingredient the synthetic antisense oligodeoxynucleotide hEN101,as denoted by SEQ ID NO:1, and optionally further comprising additionaltherapeutic agents and/or pharmaceutically acceptable carriers,excipients and/or diluents. Preferably, said pharmaceutical compositionis for the treatment and/or prevention of myasthenia gravis.

[0130] The progressive neuromuscular disorder to be treated and/orprevented by the pharmaceutical composition of the invention isassociated with an excess of AChE or protein. Usually, said excessiveAChE will be the AChE-R variant or isoform.

[0131] In addition, said progressive neuromuscular disorder to betreated and/or prevented by the pharmaceutical composition of theinvention is associated with impairment of the cholinergic transmission.Said disorder may involve muscle distortion, muscle re-innervation, orneuromuscular junction (NMJ) abnormalities.

[0132] The pharmaceutical composition of the invention is for use in thetreatment and/or prevention of a disorder such as myasthenia gravis(MG), Eaton-Lambert disease, muscular dystrophy, amyotrophic lateralsclerosis (ALS), post-traumatic stress disorder (PTSD), multiplesclerosis (MS), dystonia, post-stroke sclerosis, post-injury muscledamage, excessive re-innervation, post-surgery paralysis of unknownorigin and post-exposure to AChE inhibitors.

[0133] In one embodiment, the pharmaceutical composition of theinvention is for daily use by a patient in need of such treatment, at adosage of active ingredient between about 0.001 μg/g and about 50 μg/g.Preferably, the treatment and/or prevention comprises administering adosage of active ingredient of about 0.01 to about 5.0 μg/g. Mostpreferably, said dosage of active ingredient is of between about 0.05 toabout 0.70 μg/g, and even most preferably, the dosage is from 0.15 to0.50 μg/g of body weight of the patient.

[0134] As may be seen in Example 11 and FIGS. 18-21, treatment of MGpatients with hEN101 resulted in significant improvement of the clinicalsymptoms. As an example of such improvements, FIG. 18 shows how patientswere capable of better opening their eyes (lifting their eye lids), andtheir QMG scores improved significantly (FIGS. 19-21). Thus, hEN101 hasproved to be effective in reversing the symptoms in human MG patients.

[0135] The pharmaceutical composition of the invention may optionallycomprise at least one additional active agent. Said active agent may be,for example, AChE inhibitors used for the treatment of neuromusculardisorders.

[0136] In a further aspect, the invention relates to a pharmaceuticalcomposition comprising an antisense oligodeoxynucleotide as denoted bySEQ ID NO:1, for facilitating passage of compounds through the BBB,optionally further comprising additional pharmaceutically active agentand/or pharmaceutically acceptable adjuvant, carrier or diluent. Theadditional pharmaceutically active agent is a compound to be transportedthrough the BBB. These pharmaceutical compositions of the invention maybe used for treatment of disorders associated with the central nervoussystem, particularly such disorders that require administration of anactive agent into the CNS, for example, for the treatment of braintumors. Conventional chemotherapeutic agents do not pass the BBB, andare therefore ineffective [de Angelis, L. M., N. Engl. J Med. 433,114-123 (2001)]. As the antisense oligonucleotide of the invention hasbeen shown to penetrate the BBB, brain tumors could be treated byinjection or oral administration of the antisense oligonucleotide of theinvention, preventing or reducing the need for methods requiringinvasion of the CNS. Antisense oligonucleotides can be madetumor-specific [Ratajczak M. Z. et al. Proc. Natl. Acad. Sci. USA 89,11823-11827 (1992)]; therefore should they be found to pass the BBB,they may be both specific and effective. Thus, the additionalpharmaceutical agents comprised in these compositions of the inventionmay be, for example carcinostatic and metastatic drugs.

[0137] A number of compounds are needed for the diagnostic or treatmentof conditions affecting the central nervous system, wherein the BBBwould normally impede their delivery. These conditions can include anydisease or pathology, which include but are not limited to infections,neurochemical disorders, brain tumors and gliomas, demyelination, otherneuropathies, encephlopathies, coma, ischemia, hypoxia, epilepsy,dementias, cognitive disorders, neuropsychiatric disorders (as forexample depression, anxiety, schizofrenia and the like), as well asgenetic disorders. Thus, said compounds or additional pharmaceuticallyactive agent to be transported across the BBB may be, for example,contrast agents (dyes) used for central nervous system imaging, drugssuch as antibiotics or chemotherapeutics, gene therapy vectors, or evenagents that function to block the effects of abused drugs. Theadministration and dosage of these compounds shall be according to whatis known in medical practice, which generally should take into accountthe clinical condition of the patient in need of such treatment, as wellas said patient's age, sex, body weight and other factors known to beimportant in the medical practice. The site and method of administrationshould also be chosen accordingly. The pharmaceutically effective amountfor purposes herein is thus determined by such considerations as areknown in the art. The compound can be administered in several ways asdescribed for the delivery of the composition (see below).

[0138] The compound to be transported through the BBB may beadministered simultaneously with the composition of the invention or canbe administered at some point during the biologically effective periodof the action of the composition. In other words, the composition of theinvention facilitates the disruption of the BBB, i.e. it opens the BBB,for a period of time depending on its dose and the compound can then beadministered during this “open” period.

[0139] In order to be effective, the antisense oligonucleotide of theinvention, also when comprised in a pharmaceutical composition of theinvention, must travel across cell membranes. In general, antisenseoligonucleotides have the ability to cross cell membranes, apparently bya saturable uptake mechanism linked to specific receptors. As antisenseoligonucleotides are single-stranded molecules, they are to a degreehydrophobic, which enhances passive diffusion through membranes.Modifications may be introduced to an antisense oligonucleotide toimprove its ability to cross membranes. For instance, theoligonucleotide molecule may be linked to a group comprising optionallypartially unsaturated aliphatic hydrocarbon chain and one or more polaror charged groups such as carboxylic acid groups, ester groups, andalcohol groups. Alternatively, oligonucleotides may be linked to peptidestructures, which are preferably membranotropic peptides. Such modifiedoligonucleotides penetrate membranes more easily, which is critical fortheir function and may therefore significantly enhance their activity.Palmityl-linked oligonucleotides have been described by Gerster et al.[Anal. Biochem. 262, 177-84 (1998)]. Geraniol-linked oligonucleotideshave been described by Shoji et al. [J. Drug Target 5, 261-73 (1998)].Oligonucleotides linked to peptides, e.g., membranotropic peptides, andtheir preparation have been described by Soukchareun et al. [Bioconjug.Chem. 9, 466-75 (1998)]. Modifications of antisense molecules or otherdrugs that target the molecule to certain cells and enhance uptake ofthe oligonucleotide by said cells are described by Wang, J. [ControlledRelease 53, 39-48 (1998)].

[0140] Any of the compositions of the invention are for use byinjection, topical administration or oral uptake. Preferred uses of thepharmaceutical compositions of the invention by injection aresubcutaneous injection, intraperitoneal injection, and intramuscularinjection. As shown in the following Examples, oral administrationproved very effective, it is much easier to prescribe and meet patientcompliance, and it involves more easy handling.

[0141] The compositions of the present invention suitable for oraladministration may be presented as discrete units such as capsules,sachets or tablets, each containing a predetermined amount of the activeingredient; as a powder or granules; as a solution or suspension in anaqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. A tablet may be made bycompression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with a binder (e.g., povidone,gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent,preservative, disintegrant (e.g., sodium starch glycolate, cross-linkedpovidone, cross-linked sodium carboxymethyl cellulose) surface-active ordispersing agent. Molded tablets may be made by molding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein using, for example, hydroxypropylmethyl cellulose invarying proportions to provide the desired release profile. Tablets mayoptionally be provided with an enteric coating, to provide release inparts of the gut other than the stomach.

[0142] The pharmaceutical compositions of the invention generallycomprise a buffering agent, an agent which adjusts the osmolaritythereof, and optionally, one or more carriers, excipients and/oradditives as known in the art, e.g., for the purposes of adding flavors,colors, lubrication, or the like to the pharmaceutical composition. Apreferred buffering agent is phosphate-buffered saline solution (PBS),which solution is also adjusted for osmolarity.

[0143] Carriers may include starch and derivatives thereof, celluloseand derivatives thereof, e.g., microcrystalline cellulose, xantham gum,and the like. Lubricants may include hydrogenated castor oil and thelike.

[0144] A preferred pharmaceutical formulation is one lacking a carrier.Such formulations are preferably used for administration by injection,including intravenous injection.

[0145] The preparation of pharmaceutical compositions is well known inthe art and has been described in many articles and textbooks, see e.g.,Remington's Pharmaceutical Sciences, Gennaro A. R. ed., Mack PublishingCo., Easton, Pa., 1990, and especially pp. 1521-1712 therein.

[0146] Additives may also be designed to enhance uptake of the antisenseoligonucleotide across cell membranes. Such agents are generally agentsthat will enhance cellular uptake of double-stranded DNA molecules. Forinstance, certain lipid molecules have been developed for this purpose,including the transfection reagents DOTAP (Roche Diagnostics),Lipofectin, Lipofectam, and Transfectam, which are availablecommercially. For a comparison of various of these reagents in enhancingantisense oligonucleotide uptake see e.g., Quattrone et al. [Biochemica1, 25, (1995)] and Capaccioli et al. [Biochem. Biophys. Res. Comm. 197,818 (1993). The antisense oligonucleotide of the invention may also beenclosed within liposomes. The preparation and use of liposomes, e.g.,using the above mentioned transfection reagents, is well known in theart. Other methods of obtaining liposomes include the use of Sendaivirus or of other viruses. Examples of publications disclosingoligonucleotide transfer into cells using the liposome technique aree.g., Meyer et al. [J. Biol. Chem. 273, 15621-7 (1998)], Kita and Saito[Int. J. Cancer 80, 553-8 (1999)], Nakamura et al. [Gene Ther. 5,1455-61 (1998)] Abe et al. [Antivir. Chem. Chemother. 9, 253-62 (1998)],Soni et al. [Hepatology, 28, 1402-10 (1998)], Bai et al. [Ann. Thorac.Surg. 66, 814-9 (1998) and see also discussion in the same journal p.819-20], Bochot et al. [Pharm. Res. 15, 1364-9 (1998)], Noguchi et al.[FEBS Lett. 433, 169-73 (1998)], Yang et al. [Circ. Res. 83, 552-9(1998)], Kanamaru et al. [J. Drug Target. 5, 235-46 (1998)] andreferences therein. The use of Lipofectin in liposome-mediatedoligonucleotide uptake is described in Sugawa et al. [J. Neurooncol. 39,237-44 (1998)]. The use of fusogenic cationic-lipid-reconstitutedinfluenza virus envelopes (cationic virosomes) is described in Waelti etal. [Int. J. Cancer, 77, 728-33 (1998)].

[0147] The above-mentioned cationic or nonionic lipid agents not onlyserve to enhance uptake of oligonucleotides into cells, but also improvethe stability of oligonucleotides that have been taken up by the cell.

[0148] The invention also relates to a method for the treatment orprevention of a progressive neuromuscular disorder or other diseaseinvolving excessive production of AChE-R mRNA, comprising administeringthe oligodeoxynucleotide of the invention or a pharmaceuticalcomposition of the invention or of any of the preferred embodimentsthereof, to a patient in need thereof.

[0149] Lastly, the invention relates to a method of administering to apatient in need of such treatment a therapeutic agent for treatment of adisorder or disease of the CNS, comprising the steps of administering tosaid patient the antisense oligodeoxynucleotide of the invention andsaid therapeutic agent. The administration of the therapeutic agent maybe simultaneous with the administration of that of the antisenseoligodeoxynucleotide of the invention, or preceding or following thesame. Rupture of the BBB by the antisense oligodeoxynucleotide of theinvention will facilitate the passage of the therapeutic agent acrossthe BBB and into the CNS, where its effect is required.

[0150] Disclosed and described, it is to be understood that thisinvention is not limited to the particular examples, process steps, andmaterials disclosed herein as such process steps and materials may varysomewhat. It is also to be understood that the terminology used hereinis used for the purpose of describing particular embodiments only andnot intended to be limiting since the scope of the present inventionwill be limited only by the appended claims and equivalents thereof.

[0151] It must be noted that, as used in this specification and theappended claims, the singular forms “a”, “an” and “the” include pluralreferents unless the content clearly dictates otherwise.

[0152] Throughout this specification and the claims which follow, unlessthe context requires otherwise, the word “comprise”, and variations suchas “comprises” and “comprising”, will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

[0153] The following Examples are representative of techniques employedby the inventors in carrying out aspects of the present invention. Itshould be appreciated that while these techniques are exemplary ofpreferred embodiments for the practice of the invention, those of skillin the art, in light of the present disclosure, will recognize thatnumerous modifications can be made without departing from the spirit andintended scope of the invention.

EXAMPLES Experimental Procedures

[0154] Animals:

[0155] Rats: EAMG was induced in female Lewis rats (120-150 g) purchasedfrom the Jackson Laboratory (Bar Harbor, Me.), and housed in the AnimalFacility at the Hebrew University Faculty of Medicine, in accordancewith NIH guidelines. Control FVB/N mice were subjected to confined swimstress as described [Kaufer et al., 1998 id ibid.]. Transgenic FVB/Nmice overexpressing AChE-R were as detailed [Sternfeld et al. (2000)Proc. Natl.Acad. Sci. USA 97, 8647-8652].

[0156] Monkeys. Purpose-bred female and male 15 month-old Cynomolgusmonkeys were used.

[0157] Oligonucleotides: HPLC-purified, GLP grade oligonucleotides(purity>90% as verified by capillary electrophoresis) were purchasedfrom Hybridon, Inc. (Worchester, USA). Lyophilized oligonucleotides wereresuspended in sterile double distilled water (24 mg/ml), and stored at−20° C. The oligonucleotides were prepared with phosphodiester linkagesat all but the three terminal 3′ positions at which 2′-O-methylribonucleotide substitutions were made. The primary sequences used inthis study were: hEN101 5′-CTGCCACGTTCTCCTGCACC-3′ (human AS3, SEQ IDNO:1) 2′-O-methylated hEN101 (methylated nucleotides marked with *)5′-CTGCCACGTTCTCCTGCA*C*C*-3′ mEN101 5′-CTGCAATATTTTCTTGCACC-3′ (mouseAS3, SEQ ID NO:3) [Grifman, M., and Soreq, H. (1997) Antisense NucleicAcid Drug Dev 7, 351-9] rEN101 5′-CTGCGATATTTTCTTGTACC-3′ (rat AS3, SEQID NO:4) [WO98/26062] rEN102 5′-GGGAGAGGAGGAGGAAGAGG-3′ (SEQ ID NO:5)[WO98/26062] r-invEN102 5′-GGAGAAGGAGGAGGAGAGGG-3′ (SEQ ID NO:6)[Meshorer, E. et al. (2002) id ibid]

[0158] Stability of hEN101: hEN101 was found to be stable (>90% oforiginal concentration) after storage for 2 h in human plasma with EDTAat room temperature, following three freeze/thawing cycles, or after 1month at −20° C. Exposure at room temperature to Li-heparin-treatedblood caused a decay of hEN101 with a half-life in the order of 30 min.

[0159] Antibodies: Rabbit polyclonal antibodies against the C-terminalAChE-R were prepared and purified as described [Sternfeld et al. (2000)ibid.]. Goat polyclonal anti-AChR (C-20, S.C.-1448) antibodies were fromSanta Cruz, (Santa Cruz, Calif.).

[0160] Induction of EAMG: Torpedo acetylcholine receptor (T-AChR) waspurified from T. californica electroplax by affinity chromatography onneurotoxin-Sepharose resin, as previously described [Boneva, N. et al.(2000) Muscle & Nerve 23, 1204-8]. Rats were immunized with 40 μg ofpurified T-AChR emulsified in complete Freund's adjuvant supplementedwith 1 mg of M. tuberculosis H37Ra (Difco, Detroit Mich.). The animalswere injected subcutaneously in the hind footpads and a boosterinjection of the same amount was given after 30 days. A third injectionwas administered to animals that did not develop EAMG after the secondinjection. Animals were weighed and inspected weekly during the firstmonth and daily after the booster immunization, for evaluation of muscleweakness. The clinical status of the rats was graded according to:0—Without definite weakness (treadmill running time, 23±3 min); mild(1)—weight loss>3% during a week, >10 min. running time on treadmill;moderate (2)—moderate weakness accompanied by weak grip or cry withfatigue, weight loss of 5-10%, 3-5 min. running on treadmill;moderate-severe (3)—moderate to severe weakness, hunched back posture atrest, head down and forelimb digit flexed, tremulous ambulation, 10%body weight loss, 1-2 min. run on treadmill); severe (4)—severe generalweakness, no cry or grip, treadmill running time<1 min, weight loss>10%;(5)—death.

[0161] Anti-AChR antibody determination: Serum was assayed by directradioimmunoassay, using ¹²⁵I-α bungarotoxin (BgT) bound to T-AChR and torat (R) AChR [Boneva et al. (2000) id ibid]. All the EAMG rats displayedhigh anti-T-AChR or anti-R-AChR titers, with serum mean±standard error(SE) values of 82.1±16.0 nM for anti-T-AChR antibodies and 19.9±1.8 nMfor anti-R-AChR. Human serum was tested for the level of anti-AChRantibodies as previously described [Drachman, D. B. (1994) N Engl J Med330, 1797-810].

[0162] Quantification of nAChR: AChR concentration in the gastrocnemiusand tibialis muscles was determined using ¹²⁵I-α-BgT binding followed byprecipitation by saturated ammonium sulfate as described previously[Boneva et al. (2000) id ibid].

[0163] Immunocytochemistry: Muscle sections were deparaffinized withxylene and were re-hydrated in graded ethanol solutions (100%, 90%, 70%)and PBS. Heat-induced antigen retrieval was performed by microwavetreatment (850 W for rapid boil following 10 min in reduced intensity)in 500 ml of 0.01M citrate buffer pH 6.0. Slides were cooled to roomtemperature and rinsed in double distilled water. Non-specific bindingwas blocked by 4% normal donkey serum in PBS with 0.3% Triton X-100 and0.05% Tween 20 (1 hr at room temperature). Biotinylated primary antibodywas diluted (1:100 and 1:30 for rabbit anti-AChE-R [Sternfeld et al.(2000) id ibid] and goat anti-nAChR, respectively) in the same bufferand slides were incubated 1 hr at room temperature following overnightincubation at 4° C. Sections were rinsed and incubated with alkalinephosphatase-conjugated secondary antibody, diluted in the same blockingbuffer 1 hr at room temperature and then overnight at 4° C. Detectionwas with the alkaline phosphatase substrate Fast Red (Roche Diagnostics,Mannheim, Germany). Slides were simultaneously transferred to a stopsolution (25 mM EDTA, 0.05% Triton X-100, 1 mM levamisole in PBS, pH7.2), rinsed in PBS and cover-slipped with Immunomount (Shandon).

[0164] For spinal cord sections, primary mouse anti-SC35 antibody wasdiluted (1:100) in the same buffer as the previous primary antibodies,and slides were incubated 1 h at room temperature following overnightincubation at 4° C. Sections were rinsed and incubated with peroxidaseconjugated goat anti-mouse secondary antibody, diluted in the sameblocking buffer, for 1 h at room temperature and then overnight at 4° C.Detection was performed with DAB substrate (Sigma). Slides werecover-slipped with Immunomount (Shandon, Pittsburgh, Pa.).

[0165] Electromyography: Rats were anesthetized by i.p. injection of 2.5mg/Kg pentobarbital, immobilized, and subjected to repetitive sciaticnerve stimulation, using a pair of concentric needle electrodes at 3 Hz.Baseline compound muscle action potential (CMAP) was recorded by aconcentric needle electrode placed in the gastrocnemius muscle,following a train of repetitive nerve stimulations at supramaximalintensity. Decrease (percent) in the amplitude of the fifth vs. thefirst muscle action potential was determined in two sets of repetitivestimulations for each animal. A reduction of 10% or more was consideredindicative of neuromuscular transmission dysfunction.

[0166] Drug administration: Intravenous injections and blood samplingfor anti-AChR antibodies testing were via the right jugular vein underanesthesia. For oral administration, a special needle for oral gavagefeeding was used, which is curved with a ball end (Stoelting, Wood DaleIll.). Mestinon®) was administered in a dose of 1 mg/kg/day, andpurchased from Hoffmann La-Roche, Basel, Switzerland.

[0167] Exercise training on treadmill: To establish a clinical measureof neuromuscular performance in EAMG rats, a treadmill assay wasperformed. Animals were placed on an electrically powered treadmill[Moran et al. (1996) J Therm Biol 21, 171-181] at 25 m/min (a physicaleffort of moderate intensity) until visibly fatigued. The amount of timethe rats were able to run was recorded before and after anti-sense orMestinon® treatment.

[0168] In situ hybridization: Tissues were fixed in 4% paraformaldehydeand cut into 7 μm paraffin embedded sections. Spinal cord sections weredeparaffinized, rehydrated using serial ethanol dilutions andpermeabilized with proteinase K (10 μg/ml at room temp.). Slides wereexposed to 5′ biotinylated, fully 2′-oxymethylated AChE-R or AChE-S-specific 50-mer cRNA probes complementary to human ACHE pseudo-intron 4or exon 6, respectively [Grisaru et al. (2001) id ibid.]. Hybridizationwas performed overnight at 52° C. in hybridization mixture containing 10μg/ml probe, 50 μg/ml yeast tRNA, 50 μg/ml heparin and 50% formamide in375 mM Na chloride, 37.5 mM Na citrate, pH 4.5. For monkey sections, theprobe was constructed according to the human AChE-R sequence; for ratsections, according to the mouse sequence. Slides were washed to removenon-hybridized probe, blocked with 1% skim milk containing 0.01 %Tween-20 and 2 mM levamisole, an alkaline phosphatase inhibitor used tosuppress non-specific staining and incubate with streptavidin-alkalinephosphatase (Amersham Pharmacia). Fast Red™ substrate (RocheDiagnostics) was used for detection. DAPI staining (Sigma Chemical Co.,St. Louis, Mo., USA) served to visualize nuclei. Microscope images wereanalyzed with Image Pro Plus 4.0 (Media Cybernetics) software.

[0169] Serum analyses: Blood samples drawn from EAMG rats and MGpatients were subjected to non-denaturing gel electrophoresis asdescribed [Kaufer et al. (1998) id ibid], as well as to catalyticactivity measurements of AChE [Shapira et al. (2000) id ibid]. Iso-OMPA(tetraisopropylpyrophosphoramide, 5×10⁻⁵ μM), was used to blockbutyrylcholinesterase activity in the serum samples. For activitystaining on polyacrylamide gels [Kaufer et al. (1998) id ibid] we used510-6 M iso-OMPA.

[0170] Protocol for Phase Ib Clinical Trial of MG patients using hEN101:Patients were hospitalized and pyridostigmine was discontinued for 12-18hours before EN101 testing. Assessment of MG status was performed firstat entry, then after pyridostigmine stoppage, and regularly after EN101treatment using a Quantitative MG (QMG) score. Escalating oral doses ofEN101 (10-150 μg/kg) were given in the first day (day 1), followed by adaily dose of 500 μg/kg for 3 days (days 2-4). Days 5 and 6 were washoutperiod without pyridostigmine, and restitution of pyridostigmineoccurred when it became necessary. Patients were monitored for 1 monththereafter, with three visits as out-patients.

[0171] The following parameters were used as inclusion criteria:

[0172] Class II and above, according to MGFA classification (myastheniagravis standard clinical classification of the disease severity score);

[0173] Age 18-70;

[0174] Seropositive for AChR antibodies;

[0175] Patients under pyridostigmine (180 mg/day) treatment withoutconcomitant immunosuppressants;

[0176] Stable for 3 months, with no PE (plasma exchange) for 6 months;

[0177] No other major or active diseases.

[0178] Evaluation of the MG status of each patient was based on thefollowing parameters:

[0179] (a) QMG scoring (maximum value of 9), based on the measurementsof:

[0180] Fatigue in each limb (4);

[0181] Fatigue of the neck (1);

[0182] Swallowing rate (1);

[0183] Power in the hands (2);

[0184] Respirometry (1).

[0185] These measurements were taken daily, 4 times per day, andaveraged for days 2-6.

[0186] (b) Patient's subjective report;

[0187] (c) Vital signs, clinical chemistry, hematology, urinalysis, ECGand physical examination, recorded daily.

EXAMPLE 1 AChE-R Accumulate in Blood and Muscle of EAMG Rats

[0188] As previously shown by the inventors, the AChE-R variant migrateson non-denaturing polyacrylamide gels faster than the tetramericsynaptic enzyme, AChE-S [Kaufer et al. (1998) id ibid.], and it ispresent in the serum of MG patients [WO01/36627]. Similarly, immunoblotanalysis confirmed that in EAMG rats, as compared with healthy rats,there was a massive increase in serum AChE-R (FIG. 7).

[0189] Expression of alternative AChE variants (FIG. 8A), as well as ofthe nicotinic acetylcholine receptor (nAChR), was tested in control andEAMG rats. Depletion of nAChR in muscle sections from EAMG rats wasdetected, as evidenced by a quantitative immunoassay using antibodiesagainst nAChR (FIG. 8B, 1 and 2). The immunostaining showed that musclenAChR was reduced by 48±7% from normal values in 10 mildly affectedanimals (disease grade 1-2, see Experimental Procedures) and by 75±5% in10 severely affected rats (grade 4) compared to controls (FIG. 8B, 1 and2), attesting to the myasthenic nature of this animal model.Immunohistochemical staining with a polyclonal antiserum thatselectively detects AChE-R [Sternfeld et al. (2000) id ibid] revealedpositive signals in some, but not all muscle fibers of control rats.Similar patterns appeared under treatment with the inert, inverselyoriented oligonucleotide r-invEN102 (see FIG. 8B, 3). In EAMG rats (FIG.8B, 4), staining of AChE-R showed it as more generally distributed, withthe dispersed cytoplasmic localization that is characteristic of thisisoform [Soreq, H., and Seidman, S. (2001) Reviews Neuroscience 2,294-302], contrasting with the sub-synaptic cluster distribution of thesynaptic variant [Rossi, S. G. and Rotundo, R. L. (1993) J Biol Chem268, 19152-9]. Both the level of expression and the cellulardistribution of muscle AChE-S were similar in EAMG and healthy,untreated and r-invEN102-treated rats.

[0190] In situ hybridization using variant-selective probes showed thatAChE-S mRNA was sub-synaptically located in muscles from both untreatedand r-invEN102-treated, healthy and EAMG rats (FIG. 8B, 5 and 6). Incontrast, healthy rats displayed weaker and diffuse labeling of theAChE-R mRNA transcript, whereas a more pronounced punctuate labeling ofAChE-R mRNA appeared in triceps muscles of EAMG rats, unaffected byr-invEN102 treatment (FIG. 8B, 7 and 8). This accumulation in regionsrich in densely clustered nuclei was consistent with previousobservations of sub-synaptic regions [Rossi and Rotundo (1993) id ibid].These data indicate a selective over-expression of AChE-R in muscles ofEAMG rats and strengthened the idea of a role for this enzyme variant inMG pathophysiology.

EXAMPLE 2 AChE-R and AChE-R mRNA Levels in Muscle Respond to rEN101

[0191] The soluble and secretory nature of AChE-R predicted that itwould degrade acetylcholine before it reaches the post-synapticmembrane, limiting receptor activation. To test this hypothesis, rEN101antisense oligonucleotide was used, which is capable of selectivesuppression of AChE-R production [Galyam, N. et al. (2001) AntisenseNucl Acid Drug Dev 11, 51-57]. AChE-R suppression was tested in healthyand EAMG rats with reduced muscle nAChR levels (FIG. 8B, 1 and 2) 24 hafter a single i.v. injection of 250 g/Kg rEN101. Immunohistochemicalstaining demonstrated that AChE-R, but not AChE-S, was significantlyreduced in muscles from both healthy and EAMG rats (FIG. 8C, 3 and 4 anddata not shown). Receptor labeling patterns remained high in healthyrats and low in EAMG animals, similar to those of untreated animals andanimals treated with r-invEN102 (compare FIG. 8B, 1 and 2 to FIG. 8C, 1and 2). In situ hybridization indicated that AChE-S mRNA labeling,limited to the sites of subsynaptic clusters of nuclei, was onlynominally affected by rEN101, suggesting that neuromuscular transmissionwould be unaffected by this treatment (FIG. 8C, 5 and 6). In contrast,rEN101 reduced AChE-R mRNA labeling almost to the limit of detection inboth healthy and myasthenic rats (FIG. 8C, 7 and 8).

EXAMPLE 3 Suppression of AChE-R Restores Normal CMAP in EAMG Rats

[0192] Quantification by densitometry of an immunoblot analysisconfirmed the increase of serum AChE-R in EAMG and the efficacy of asingle i.v. injection of 250 μg/Kg rEN101, but not r-invEN102, inreducing its serum level 24 h later (FIG. 9A). To evaluate thephysiological outcome of this suppression, compound muscle actionpotentials (CMAPs) from the gastrocnemius muscle were recorded. EAMGrats, but never healthy animals, displayed a decline in CMAP duringrepeated stimulation at 3 Hz. The baseline decline, the percentdifference in the heights of the fifth and the first evoked potentials,ranged from 10% to 36% (mean±SEM=13.0±2.5%, FIG. 9B, inset) as comparedto 4.0±0.9% among healthy rats. The standard therapy for MG patients isadministration of anti-cholinesterases, which elevate ACh levels to athreshold that enables receptor activation. Accordingly, neostigminebromide (Prostigmine™, 75 μg/kg) was administered via i.p. This rapidlyand effectively corrected the CMAP decline in EAMG rats, from 87.6% ofthe first evoked potential in untreated animals to over 120% of thislevel (i.e. 107.4%) of the first evoked potential). The effects of thecholinesterase blockade were evident starting 15 min after the injectionand lasted 2 h, after which time the CMAP value returned to the baseline(FIG. 9B).

[0193] Unlike anticholinesterases, which block all AChE variants, rEN101was shown to selectively suppress muscle AChE-R production [Lev-Lehmanet al. (2000) id ibid]. Therefore, retrieval of stable CMAP inrEN101-teated EAMG rats may attest to the causal role of AChE-R in theneuromuscular malfunctioning that is characteristic of the myasthenicphenotype. To test this concept, rEN101 was injected i.v. at dosesranging from 10-500 μg/Kg (2 to 20 nmol/rat). rEN101 did not affect CMAPin healthy animals, but retrieved stable CMAP ratios within 1 h (FIGS.9B, inset, 9C and Table 1). CMAP normalization was accompanied byincreased mobility, upright posture, stronger grip, and reducedtremulousness of ambulation. TABLE 1 Post-treatment CMAP ratios^(a)Oral^(b) Intravenous^(b) Phenotype Naive EAMG EAMG EAMG EAMG Naive EAMGEAMG EAMG Treatment EN101 EN101 EN102 invEN102 pyridostigmine EN101EN101 EN102 invEN102 0 h 1.01 ± 0.01 0.84 ± 0.03 0.82 ± 0.02 0.78 ± 0.060.90 ± 0.01 1.00 ± 0.0 0.87 ± 0.01 0.85 ± 0.06 0.89 ± 0.02 (4) (8) (4)(4) (6) (6) (6) (4) (5) 1 h^(c) 1.0 ± 0.02 0.97 ± 0.02 0.86 ± 0.04 0.86± 0.05 0.98 ± 0.01 0.02 ± 0.01 1.00 ± 0.01 1.04 ± 0.01 0.89 ± 0.03 (4)(8) (3) (4) (6) (7) (4) (4) (5) 5 h 1.03 ± 0.02 0.97 ± 0.03 0.96 ± 0.020.86 ± 0.05 0.96 ± 0.02 1.00 ± 0.01 0.98 ± 0.02 0.98 ± 0.03 0.89 ± 0.02(4) (7) (4) (4) (6) (6) (4) (4) (5) 24 h 1.01 ± 0.00 1.01 ± 0.01 0.95 ±0.03 0.81 ± 0.08 0.87 ± 0.02 1.02 ± 0.01 1.00 ± 0.00 1.00 ± 0.01 0.90 ±0.02 (7) (6) (5) (4) (6) (6) (4) (4) (5)

[0194] Both the extent and the duration of CMAP correction were dosedependent. For example, 500 μg/Kg conferred 72 h rectification of CMAPup to 125% of baseline, while 50 μg/Kg was effective for only 24 h.rEN102, a 3′ protected AS-ON targeting a sequence unique to rAChE-R mRNA(previously referred to as AS1) [Grifman and Soreq (1997) id ibid],induced similar rectification of CMAP decline in EAMG rats, confirmingthe relevance of AChE-R as a contributing element to this effect.Comparable amounts of r-invEN102, did not improve muscle function,attesting to the sequence specificity of the AS-ON treatment (Table 1).Dose response curves revealed that up to 5 h following an injection,rEN101 produced a saturable response with IC₅₀ of <10 μg/Kg. This effectappeared to be superimposed on a longer lasting and lessconcentration-dependent effect, which showed no saturation in the rangestudied (FIG. 9D). This phenomenon possibly reflected the altered muscleand/or neuromuscular junction properties under the stable CMAP retrievalafforded by rEN101.

EXAMPLE 4 Antisense Prevention of AChE-R Accumulation Promotes Staminain EAMG Rats

[0195] Placed on a treadmill at 25 m/min, healthy rats ran for 23.0±3.0min, after which time they displayed visible signs of fatigue. Startingat 5 h, and for at least 24 h following administration of 250 μg/KgrEN101, EAMG rats demonstrated improved performance on the treadmill.Running time increased from 247±35, 179±21 and 32 ±6 sec to 488±58,500±193 and 212±59 sec for animals at disease grades 2, 3 and 4,respectively (average values for 6-9 animals per group.) Healthyanimals, in contrast, were not significantly affected by rEN101injection (FIG. 10).

[0196] Others have demonstrated efficacy of orally administered2′-oxymethyl protected AS-ON agents [Monia, B. P. (1997) Ciba Found.Symp. 209, 107-119]. Therefore, the inventors tested this mode in theEAMG model. Based on their own findings, the inventors selected the doseof 50 μg/Kg of rEN101, which was administered to EAMG rats once a dayvia oral gavage, and CMAP was measured 1, 5, and 24 h later. This dosewas as effective as 25 μg/Kg administered i.v. (Table 1 and FIGS. 9 and13). Orally administered rEN102 was also active in reversing CMAPdecline, but its effects appeared somewhat delayed compared to rEN101.Oral pyridostigmine (1000 μg/kg) restored CMAP for up to several hours,while r-invEN102 had no significant effect (Table 1).

EXAMPLE 5 Oral Administration of Human EN101 to EAMG Rats

[0197] Human EN101 (hEN101) (0.25 μg/g, single dose) was administeredorally to rats with EAMG of medium severity (score 2.5-3.5), whichimplied the symptoms defined as “moderate” hereunder. The results aresummarized in Table 1. Time from treatment is noted above; together withthe treadmill running time in sec. Animals were inspected at each timepoint for evaluation of muscle weakness. The clinical status of the ratswas graded according to: (0)—Without definite weakness (treadmillrunning time, 23 ±3 min); Mild (1)—weight loss >3% during a week, >10min. running time on treadmill; Moderate (2)—moderate weaknessaccompanied by weak grip or cry with fatigue, weight loss of 5-10%, 3-5min. running on treadmill; Moderate-severe (3)—moderate to severeweakness, hunched back posture at rest, head down and forelimb digitflexed, tremulous ambulation, 10% body weight loss, 1-2 min. run ontreadmill); Severe (4)—severe general weakness, no cry or grip,treadmill running time <1 min, weight loss>10%; Death (5). Each linerepresents an individual rat. It is to be noted that the clinical score(in parentheses) was reduced in all of the treated animals, whichreflects time improvement, and that running time was significantlyincreased for over 5 and 24 hr for most of the animals and for two ofthe tested animals also at 48 h. TABLE 2 Treadmill Performance Time(Clinical score) before Animal (basal) 5 h 24 h 48 h Effect 1 110 sec150 sec 360 sec ND + + (3) (1) 2 0 (3.5)  70 sec 30 sec ND + − (3) 3 210sec 300 sec 345 sec ND + + (2.5) (1) 4 80 (3) ND 170 sec 85 (2.5) + −(2) 5 180 |||| 380 290 (2) + + (2.5) (1) 6 30 120 ||||(3) ||(5) + (3.5)

[0198] These results show that like the rat EN101 (see treadmillexample, above), the human EN101 antisense oligodeoxynucleotide of theinvention promoted muscle stamina in EAMG induced rats.

EXAMPLE 6 Comparative Analysis of hEN101 and rEN101 in a Rat AnimalModel

[0199] A study of the potential efficacy as well as toxicity of hEN101was conducted on 4 week-old Cr1:CD rats. To groups of 12 animals (6males, 6 females) were administered 0.0 (saline only), 0.50 or 2.50μg/g/day of hEN101 by oral gavage (o.g.), 0.50 μg/g/day of rEN101 by o.g., or 0.50 μg/g/day of rEN101 or hEN101 by i.v. injection.Additionally, there was a control group that was not injected. For 7days the animals were checked for gross signs of toxicity: mortality,body weight, food consumption, ophtalmology, hematology (peripheralblood), and blood chemistry. At 7 days they were sacrificed and examinedpost mortem for macroscopic pathology and organ weight. Fixed sectionsof brain (cerebellum, cerebrum, midbrain, medulla), heart (auricular andventricular regions), kidneys (cortex, medulla, papilla regions), liver(all main lobes), lungs (two major lobes, including bronchi), lymphnodes (mandibular and mesenteric), spinal cord (transverse andlongitudinal sections at cervical, lumbar and thoracic levels), caecum,colon, duodenum, ileum, jejunum, esophagus, rectum, spleen and stomach(keratinized, glandular and antrum) were stained with hematoxylin/eosinto reveal necrosis or cell death.

[0200] Mandibular lymph nodes were examined for the effect of EN101 onAChE-R mRNA. Compared to the saline-injected control, rEN101 (oral ori.v.) or hEN101 (i.v.) were inconsistent in depressing AChE-R mRNAlevels within these lymph nodes (data not shown). In contrast, theadministration of rEN101 (oral or i.v.) or hEN101 (i.v.) did not affectthe expression of the AChE-S synaptic variant of AChE in thesemandibular lymph nodes (data not shown). Thus, the antisenseoligodeoxynucleotide of the invention may be used to suppress the AChE-Rvariant without affecting the expression of the synaptic variant, i.e.without adversely affecting cholinergic transmission.

EXAMPLE 7 AChE-R Suppression Modifies the Course of EAMG Pathophysiology

[0201] As shown in Example 5, unlike anti-cholinesterases, rEN101afforded long-term maintenance of stable CMAP. This further enabled theinventors to test whether the cholinergic imbalance contributes to thephysiological deterioration that is characteristic of EAMG. Rats werefirst treated with rEN101 once a day for 5 days, CMAPs being determinedprior to each treatment. Both the efficacy of rEN101 in retrievingnormal CMAP and its capacity to reduce the inter-animal variability inCMAP values reached similar levels to those of pyridostigmine (FIGS. 11Aand 11B). However, the onset of response to pyridostigmine was morerapid (Table 1), while that observed with rEN101 was longer-lasting.Daily oral or i.v administration of rEN101 stabilized CMAPs over theentire course of treatment (FIG. 11B). In contrast, the effect ofpyridostigmine wore off within several hours, causing pronouncedfluctuations in muscle status (Table 1 and FIG. 11). Among the animalstreated daily with pyridostigmine, 5 out of 6 died within the 5 dayexperimental course. In contrast, 6 out of 8 animals treated once-a-daywith rEN101 via o.g. survived the full 5 day period. This conspicuousdifference might reflect the susceptibility of EAMG rats to repeatedanesthesia and CMAP measurements. In order to avoid these additionalstresses and evaluate the effect of the antisense treatment on EAMGpathophysiology, the inventors subjected groups of moderately sickanimals to 1 month of daily oral treatment with minimal interference.EAMG rats receiving oral doses of rEN101 daily, presented significantimprovement in survival, clinical status and treadmill performance, ascompared with pyridostigmine- and saline-treated animals (FIG. 12;P<0.041 for 4 weeks survival incidence, Fisher exact test, AS-ON vs.other treatments). One way repeated measures ANOVA yielded P<0.05 forall other measures (AS vs. other treatments at 4 weeks). The effect ofrEN101 on clinical symptoms was also corroborated by body weightchanges. Rats treated with saline and Mestinon treated groups lost 13.5and 11 g/animal, respectively, whereas animals treated with rEN101gained, on average, 13 g during the treatment period. Thus, daily rEN101administration promoted long-term change in the course of EAMG in ratswith moderate to severe symptoms, under the same conditions in whichuntreated or pyridostigmine-treated animals deteriorated.

[0202] By using MG and EAMG as case studies for evaluating theconsequences of chronic neuromuscular imbalance at the level of geneexpression, the inventors confirmed that the AChE-R variant issystemically elevated in MG and EAMG. Moreover, the inventors showedthat antisense suppression of AChE-R normalized NMJ responses torepeated nerve stimulation, promoting muscle strength, and recuperatinga healthier status in animals otherwise too weak even to eat. Theseobservations support the idea that AChE-R plays a direct role in MGpathophysiology and call for evaluation of the rationale of long-termmRNA-targeted therapy for imbalanced cholinergic function at NMJs.

EXAMPLE 8 Oral Administration of hEN101 to Cynomolgus Monkeys

[0203] This experiment was conducted with six (3 males and 3 females)purpose-bred 15 month-old (young adult) Cynomolgus monkeys, divided inthree groups (1, 2 and 3) of one male and one female each. Groups 1 and2 received hEN101 daily by o.g. for a period of 7 days, at aconcentration of 0.15 and 0.50 μg/g/day, respectively. Group 3 receiveddaily i.v. injections of hEN101 for a period of 7 days at a dosage of0.50 μg/g/day.

[0204] Over a 12 hour period, plasma samples were obtained during thesecond treatment day to investigate the toxicokinetic profile at eachdosage. The toxicology study consisted of checking the animals duringthe 7 days of treatment for gross signs of toxicity by the followingparameters: mortality, body weight, food consumption,electrocardiography, blood pressure, hematology (peripheral blood), andblood chemistry. At 7 days the monkeys were sacrificed and examinedpost-mortem for macroscopic pathology and organ weight. Fixed sectionsof brain (cerebellum, cerebrum, midbrain, medulla), caecum, colon,duodenum, heart (auricular and ventriclar regions), ileum, jejunum,kidneys (cortex, medulla, papilla regions), liver (two main lobes),lungs (two major lobes, including bronchi), lymph nodes (mandibular andmesenteric), esophagus, rectum, sciatic nerve, skeletal muscle (thigh),spinal cord (transverse and longitudinal sections at cervical level),spleen and stomach (body and antrum) were stained with hematoxylin/eosinto reveal necrosis or cell death.

[0205] These clinical investigations revealed no toxicological effectsby any of the treatment protocols. Post-mortem there were notreatment-related effects other than slight healing erosions in the bodyof the stomach, which are possibly associated with treatment, in 1 of 2animals in each group, and some irritation of the perivascular tissue atthe site of intravenous injection.

[0206] Paraffin-embedded 7 μm spinal cord sections were examined by insitu hybridization for the levels of AChE-R and AChE-S mRNA. Under allthree regimens (oral 0.15 and 0.50, and i.v. 0.50 μg/g/day), there wasno apparent reduction in AChE-S mRNA (FIG. 14). However, there was asignificant reduction in AChE-R mRNA in increasing hEN101 daily dosefrom 0.15 (oral) to 0.50 (oral or i.v.) with i.v. dosage being moreeffective.

[0207] AChE-R-positive sections from monkey spinal cords were analyzedfor the relation between cell body diameter and percentage of AChE-Rpositive cells (FIG. 15). Cells were divided into three categoriesaccording to their body diameter, and the percentage of AChE-R-positivecells from each category was evaluated. Treatment with the lowerconcentration of EN101 (150 μg/kg/day) caused an increase in the percentof small AChE-R-positive cells (<70 μm diameter) as compared to naivemonkeys, probably due to the injection stress response, which is knownto raise AChE-R mRNA levels (Kaufer et al, 1998). Either i.v. or p.o.administration of the higher EN101 concentration (500 μg/kg/day) reducedthe percentage of AChE-R-positive neurons, compared to the lowerconcentration (p<0.05, Student's t test). The decrease was moreremarkable in small neurons (23-40 μm) than in neurons with cell bodydiameter of 40-70 μm, and no decrease was observed in large neurons (>70μm diameter) (FIG. 15). The percentage of small- (23 to 40 μm-diameter)and medium-sized (40 to 70 μm) neurons that were labeled decreasedsignificantly in moving from the lower to the higher hEN 101 oral dose,and even further with the i.v. administration. Among the larger neurons(>70 μm), there was not discernable effect of hEN101. We have yet todiscover the functional correlate of cell size that determines theefficacy of antisense suppression of AChE-R expression. This suggeststhat EN101 will prevent the stress-induced impairment in interneuronsinput to motoneurons, thus preventing paralysis—e.g. post-surgery.

EXAMPLE 9 hEN101 Suppression of AChE Activity in Monkey Plasma

[0208] In the 12 hr following the second day administration of hEN101,monkey plasma samples were collected and stored. Plasma cholinesteraseactivities were measured by spectrophotometry assessing the rate ofhydrolysis of acetylthiocholine (measured by the Ellman assay, whichquantifies the hydrolysis of acetylthiocholine) [Ellman, G. L., et al.(1961), Biochem. Pharmacol. 7, 88-95], in the absence or presence ofiso-OMPA (a selective butyrylcholinesterase, BChE, inhibitor). Totalactivity, largely due to serum BChE, was generally unchanged (FIG. 16A).When measured in the presence of 1×10⁻⁵M iso-OMPA, AChE activityincreased within the 5 hr post-injection. This increase, observed under150 μg/kg was effectively suppressed or attenuated by the higher dose of500 μg/kg hEN101, and even more effective when this dose was i.v.administered (FIG. 16B).

EXAMPLE 10 Effect of rEN101 on Expression AChE-R mRNA in Rat Spinal CordNeurons

[0209] Contrary to the effect of hEN101 on monkey spinal cord neurons,rEN101 does not suppress AChE-R mRNA in the rat spinal cord (FIG. 17),when assessed by in situ hybridization using a mouse probe. Neither thenumber of positive cells nor the staining intensity were significantlychanged. One explanation for this result would be that the blood-brainbarrier that isolates the CNS is more permeable in monkeys than rats, atleast under the chosen experimental conditions.

EXAMPLE 11 hEN101 Phase Ib Clinical Trial

[0210] 16 patients with stable generalized MG requiring constant AChEinhibitors (pyridostigmine) for daily function were recruited, afterapproval by human ethics committees of the respective hospitals involvedin the present trial (Hadassah Medical Center, Jerusalem, Israel andGreater Manchester Neurosciences Center, Hope Hospital, Salford,England).

[0211] Analysis of the results obtained from the Clinical trial showedthat in 15 out of 16 patients, the initial deterioration in MG statusafter stopping pyridostigmine was followed by a clear symptomaticimprovement due to hEN101. As an example, FIG. 18 shows the improvementin ptosis in one of the MG patients. Analysis of the mean daily QMGscores showed a continuous decrease in each of the study days (decreasein QMG means improvement in disease status) (FIGS. 21A and 21B). Thebaseline (entry) mean total score was 13.2. The score decreased for days2 through 6 in the amounts of 3.0, 4.8, 5.7, 5.5 and 6.0 (p<0.001). Themean percent improvement of total QMG for these days ranged from 27.8%to 53.4% (FIG. 20) (p<0.01). All individual test item scores, except forvital capacity and left arm out stretched time, had statisticallysignificant change from entry for days 2-6 (p<0.05). Improved QCG scoresfollowing the final dose of hEN101 were sustained for up to 72 hours. Noserious adverse effects were observed. Vital signs, chemical chemistry,hematology, urinalysis, ECG and physical examination remained unchangedthroughout the experimental period and in the month following discharge.Cholinergic side effects were not reported.

[0212] Thus, hEN101 appears to be powerfully effective in reversingsymptoms in patients with stable MG. The present study showed thathEN101 has potential advantages over conventional cholinesteraseinhibitors with respect to dosing, specificity, side-effect profile,duration of efficacy and treatment regimen.

1 6 1 20 DNA Artificial Sequence Description of Artificial Sequencehuman EN101 1 ctgccacgtt ctcctgcacc 20 2 9 DNA Artificial SequenceDescription of Artificial Sequence loop within hEN101 2 cgcgaagcg 9 3 20DNA Artificial Sequence Description of Artificial Sequencemouse EN101 3ctgcaatatt ttcttgcacc 20 4 20 DNA Artificial Sequence Description ofArtificial Sequencerat EN101 4 ctgcgatatt ttcttgtacc 20 5 20 DNAArtificial Sequence Description of Artificial Sequence rat EN102 5gggagaggag gaggaagagg 20 6 20 DNA Artificial Sequence Description ofArtificial Sequencerat invEN102 6 ggagaaggag gaggagaggg 20

1. A synthetic antisense oligodeoxynucleotide targeted against humanAChE mRNA having the nucleotide sequence: 5′ CTGCCACGTTCTCCTGCACC3′  (SEQ ID NO:1).
 2. A synthetic nuclease resistant antisenseoligodeoxynucleotide having the nucleotide sequence: 5′CTGCCACGTTCTCCTGCACC 3′  (SEQ ID NO:1).
 3. A synthetic antisenseoligodeoxynucleotide of claim 2, which is a modifiedoligodeoxynucleotide comprising partially unsaturated aliphatichydrocarbon chain and one or more polar or charged groups includingcarboxylic acid groups, ester groups, and alcohol groups.
 4. A syntheticnuclease resistant antisense oligodeoxynucleotide of claim 2 or 3,wherein at least one of the three 3′-terminus nucleotides is2′-O-methylated.
 5. A synthetic nuclease resistant antisenseoligodeoxynucleotide of claim 4, in which the last three 3′-terminusnucleotides are 2′-O-methylated.
 6. A synthetic nuclease resistantantisense oligodeoxynucleotide of claim 2, wherein at least onenucleotide is fluoridated.
 7. A synthetic nuclease resistant antisenseoligodeoxynucleotide of claim 2 or claim 3, having phosphorothioatebonds linking between at least two of the last 3′-terminus nucleotidebases.
 8. A synthetic nuclease resistant antisense oligodeoxynucleotideof claim 7, having phosphorothioate bonds linking between the last four3′-terminus nucleotide bases.
 9. A synthetic nuclease resistantantisense oligodeoxynucleotide of claim 3, having a nucleotide loopforming sequence at the 3′-terminus.
 10. A synthetic nuclease resistantantisense oligodeoxynucleotide of claim 9, wherein said loop is a9-nucleotide loop having the nucleotide sequence CGCGAAGCG (SEQ IDNO:2).
 11. A synthetic nuclease resistant antisense oligodeoxynucleotideof any one of claims 1 to 10, capable of selectively modulating humanAChE production.
 12. A synthetic nuclease resistant antisenseoligodeoxynucleotide of claim 11, capable of selectively modulatinghuman AChE production in the central nervous system.
 13. Apharmaceutical composition comprising the antisense oligonucleotidehEN101, defined by SEQ ID NO:1.
 14. The pharmaceutical composition ofclaim 13, for the treatment and/or prevention of a progressiveneuromuscular disorder, for improving stamina and/or for use in chronicmuscle fatigue.
 15. The pharmaceutical composition of claim 13 for usein treating or preventing a progressive neuromuscular disorder, whereinsaid disorder is selected from myasthenia gravis, Eaton-Lambert disease,muscular dystrophy, amyotrophic lateral sclerosis, post-traumatic stressdisorder (PTSD), multiple sclerosis, dystonia, post-stroke sclerosis,post-injury muscle damage, excessive re-innervation, post-surgeryparalysis of unknown origin, and post-exposure to AChE inhibitors. 16.The pharmaceutical composition of claim 13, for the treatment and/orprevention of myasthenia gravis.
 17. The pharmaceutical composition ofclaim 13, for use in treating or preventing a progressive neuromusculardisorder, wherein said disorder is associated with an excess of AChEmRNA or protein.
 18. The pharmaceutical composition of claim 13, for usein treating or preventing a progressive neuromuscular disorder, whereinsaid disorder is associated with an excess of AChE-R mRNA.
 19. Thepharmaceutical composition of claim 13, for use in treating orpreventing a progressive neuromuscular disorder, wherein said disorderis associated with impairment of cholinergic transmission.
 20. Thepharmaceutical composition of claim 13, for use in treating orpreventing a progressive neuromuscular disorder, wherein said disorderinvolves muscle distortion, muscle re-innervation or neuro-muscularjunction (NMJ) abnormalities.
 21. The pharmaceutical composition of anyone of claims 13-20, which is for daily use by a patient of a dosage ofactive ingredient between about 0.001 μg/g and about 50 μg/g.
 22. Thepharmaceutical composition of anyone of claims 13-20, wherein thetreatment and/or prevention comprises administering a dosage of activeingredient of about 0.01 to about 5.0 μg/g.
 23. The pharmaceuticalcomposition of any one of claims 13-20, wherein the treatment and/orprevention comprises administering a dosage of active ingredient ofabout 0.15 to about 0.50 μg/g.
 24. The pharmaceutical composition ofclaim 13, optionally comprising at least one additional active agent.25. A pharmaceutical composition comprising an antisenseoligodeoxynucleotide as denoted by SEQ ID NO:1, for facilitating passageof compounds through the BBB, optionally further comprising additionalpharmaceutically active agent to be transported through the BBB, and/orpharmaceutically acceptable adjuvant, carrier or diluent.
 26. Thepharmaceutical composition of claim 25, wherein said additionalpharmaceutically active agent is selected from any one of contrastagents used for central nervous system imaging, agents that function toblock the effects of abused drugs, antibiotics, chemotherapeutic drugsand vectors to be used in gene therapy.
 27. A method of preparation of apharmaceutical composition comprising the step of admixing the antisenseoligonucleotide hEN101, defined by SEQ ID NO:1, with a pharmaceuticallyacceptable adjuvant, carrier or diluent, and optionally with at leastone additional active agent.
 28. The method of claim 27, wherein saidcomposition is intended for the treatment and/or prevention of aprogressive neuromuscular disorder, for improving stamina and/or for usein chronic muscle fatigue.